An important method for determining the sequence of amino acids in proteins and peptides is known as the Edman degradation process. This process was first described in Edman, P., Acta Chem. Scand. 4, 283(1950). The process involves coupling the N-terminal amino acid of the protein or peptide in a basic environment to phenylisothiocyanate (PITC) to form phenylthiocarbamyl (PTC) derivative, then cleaving the PTC derivative using anhydrous strong acid, such as trifluoroacetic acid, to form an anilinothiazolinone (ATZ) derivative of the N-terminal amino acid and free peptide which is the original protein or peptide with the N-terminal amino acid residue removed, then converting the (ATZ) amino acid derivative to a phenylthiohydantoin (PTH) amino acid derivative which can be analyzed by chromatography and repeating the steps for each amino acid residue that becomes the terminal residue as a result of the cleavage step.
One problem frequently encountered is that the N-terminal residue is modified in such a way that it does not react with the Edman reagent, phenylisothiocyanate, that is that the N-terminal residue is blocked. The most frequently encountered blocked N-terminal residue is an N-acetylamino acid residue. Evidence has been presented that about 80% of the soluble proteins in mammalian cells have acetylated N-terminal amino acids. (Brown, J. L., et al, J. Biol. Chem. 251, 1009-1014, 1976).
The need to remove blocking groups to convert the N-terminal residue to free N-terminal amino acid before sequencing can begin is mentioned in Kent et al U.S. Pat. No. 4,548,904. Previous attempts to remove blocking groups have involved enzymatic or limited HCl hydrolysis.
Nakamura, S., et al, Biochem. Biophys. Res. Commun. 58, 250-256 (1974) report use of rat liver peptidase to remove N-acetyl serine from the N-terminal peptide released from thrombin by bovine Factor XIII. Kobayashi, K., et al, J. Biol. Chem. 262, 11435-11445 (1987) and Jones, W. M., et al, Biochem. Biophys. Res. Commun. 139, 244-250 (1986) report use of rat liver peptidase and a similar enzyme from human erythrocytes to split off N-acetyl serine from .alpha.-melanocyte stimulating hormone. However, the applicability of the enzyme deblocking method is limited by the restricted specificity of the enzymes.
Fordyce, A. M., et al, Biochem. Soc. Trans. 7, 721-723 (1979) and Chin, C.C.Q., et al, Bioscience Rep. 5, 847-854 (1985) report some success in removing N-acetyl groups from peptides by limited HCl hydrolysis. The disadvantage of this method is that other bonds may be split besides the bond between the terminal acetyl and the nitrogen to which it is attached thereby providing a plurality of different end groups.
It is an object herein to provide a method of deblocking amino terminal N-acetyl serine and N-acetyl threonine residues in peptides and proteins to allow sequencing, with improved specificity compared to enzyme deblocking and wherein the disadvantages of hydrolysis are minimized. A survey of N-acetylated proteins by Persson, B., et al, Eur. J. Biochem. 152, 523-527 (1985) found that of the known proteins in this class, about 41% have an N-terminal acetylated serine and another 2% have an N-terminal acetylated threonine.